Musculoskeletal disorders such as rheumatoid arthritis (RA), osteoarthritis (OA), disc degeneration (DD) and osteoporosis (OP) are the major cause of morbidity throughout the world. These diseases have a substantial influence on health and quality of life and inflict an enormous cost on health systems (Scott J C, Hochberg M C. Arthritic and other musculoskeletal diseases, In: Chronic Disease Epidemiology, Brownson R C, Remington P L and Davis J L, eds. Washington, D.C. American Public Health Association, 1993). It has been estimated that musculoskeletal diseases cost the Australian community $1.3 billion annually in direct costs and $4.2 billion in indirect cost. This represents 1.3% of GNP (Arthritis Foundation of Australia, Access Economics Pty Ltd report, 2001). Population based studies conducted in other developed countries show similar incidence and health burdens to those of Australia with for example more than 23 million Americans seeking medical treatment for arthritic disorders with costs associated with their management exceeding 70 billion dollars annually (Ruchlin H S, Elkin E B, Paget S A. Assessing cost-effectiveness analyses in rheumatoid arthritis and osteoarthritis. Arthritis C Res 1997;10:413-21). Moreover, the prevalence of OA, DD and OP is rising progressively as the life span of the peoples of developed nations increases. Indeed, it has been estimated that 20% of the population in developed countries will be actively seeking medical treatments or professional heath support for musculoskeletal disabilities by the year 2020 (Croft P. The occurrence of osteoarthritis outside Europe. Ann Rheum Dis 1996;55:661-4).
The aetiology of musculoskeletal conditions such as OA is multifactorial and although ageing is the most strongly associated risk factor, mechanical, hormonal and genetic factors all contribute to varying degrees. OA emerges as a clinical syndrome when these etiological determinants result in sufficient joint damage to cause impairment of function and the appearance of symptoms. This clinical syndrome is manifest radiologically by joint space narrowing due to loss of articular cartilage (AC) and extensive re-modelling of subchondral bone with proliferation at the joint margins (osteophytosis). In the late stages of OA, joints are characterised pathologically by extensive AC fibrillation, loss of staining for proteoglycans (PGs) and eburnation of bone at sites of high contact stress. The subchondral bone beneath those regions of OA where cartilage is fibrillated or eroded is generally sclerotic and consists of immature woven bone (Felson D T. Osteoarthritis. Rheum Dis Clin Nth Amer 1990;16:499-512).
The PGs of AC consist of a protein core to which several hundred GAG chains are covalently attached. The major GAG substituents of the PGs of AC are the Chondroitin sulfates (ChS) whose isomeric structures and their distribution are well known (Poole A R. Changes in collagen and proteoglycan of articular cartilage in arthritis. Rheumatology, 1986, 10; 316-371). Thus in adult cartilage Ch-6-S is more abundant than the corresponding 4-sulfated isomer (Ch-4-S) which predominates in AC of very young animals. Within the extracellular matrix of AC the hydrated PG complexes are entrapped in the form of macromolecular aggregates by a three dimensional network of Type II collagen fibres. This unique structural organisation of PGs, water and a fibrous collagen network which is anchored in the subchondral bone plate confers to AC the biomechanical properties of resilience necessary for normal biomechanical function (Poole A R. Changes in collagen and proteoglycan of articular cartilage in arthritis. Rheumatology, 1986, 10; 316-371).
Products of cartilage breakdown in OA and RA joints have been shown to be antigenic [Giant T T, Fülöp C, Cs-Szabó G, Buzas E, Ragasa D, Mikecz K. Mapping of arthritogenic/autoimmune epitopes of cartilage aggrecans in proteoglycan-induced arthritis. Scand J Rheumatol 1995;24:43-9, Rowley M, Tait B, Mackay I R, Cunningham T, Phillips B. Collagen antibodies in rheumatoid arthritis: Significant of antibodies to denatured collagen and their association with HLA-DR4. Arthritis Rheum 1986;29:174-84, Seibel M J, Jelsma R, Saed-Nejad F, Ratcliffe A. Variability in the immunochemical quantification of keratan sulfate in human and bovine cartilage proteoglycans. Biochem Soc Trans 1990;18(5):969-70] and when released into synovial fluid (SF) may provoke a synovial inflammation. This synovitis, once established, can alter the metabolism of resident synoviocytes, the major cellular source of synovial hyaluronan (HA) in joints. Inflammatory mediators released from local macrophage and infiltrating leukocytes can also promote increased vascular permeability and the dilution of SF by plasma fluid, thereby decreasing local HA concentration (Müller-Ladner U, Gay R E, Gay S. Structure and function of synoviocytes. In: Arthritis and Allied Conditions, Koopman W J, ed. Baltimore. Williams and Wilkins, 1997, 243). This dilution of HA coupled with a reduction in its molecular size due to abnormal synthesis by synoviocytes results in a substantial decrease in the rheological properties of SF and consequently its ability to lubricate and protect AC (Balazs E A. The physical properties of synovial fluid and the special role of hyaluronic acid. In: Disorders of the Knee, Helfet A J, ed. Philadelphia. J P Lippincott, 1982, 61-74). Macrophage of the synovium, together with the leukocytes which enter the synovial cavity due to the local inflammation, are also an abundant source of cytokines [eg interleukin-1 (IL-1)], procoagulant factors, proteinases and oxygen-derived free radicals including nitric oxide radical (NO) (Pelletier J-P, DiBattista J A, Roughley P, McCollum R, Martel-Pelletier J. Cytokines and inflammation in cartilage degradation. Rheum Dis Clin N Am 1993;19:545-68, Dean D D. Proteinase-mediated cartilage degradation in osteoarthritis. Sem Arthritis Rheum 1991;20:2-11). While much of the excess proteolytic activity released into synovial fluid is abrogated by the endogenous inhibitors present, cytokines and flee radicals can freely diffuse into cartilage and down-regulate PG and collagen synthesis by chondrocytes. These proinflammatory mediators can also initiate the production of catabolic proteinases, cytokines and free radicals such as NO—by the cartilage cells which via autocrine and paracrine pathways contribute to further AC matrix destruction (Evans C H, Watkins S C, Stefanovic-Racic M. Nitric oxide and cartilage metabolism. Methods Enzymol 1996;269:75-88).
It is clear from the above that in arthritic diseases such as OA all tissues of the joint are affected and their excessive breakdown and the concomitant elicitation of an inflammatory reaction can lead not only to the progression of the disease state but also the initiation of symptoms the most common being pain and impairment of joint function.
Pharmacological management of rheumatic disorders and back pain of discal origin, has up until quite recently, targeted the symptoms of these diseases rather than the underlying pathologies which are the cause of the symptoms. Analgesics, steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) have over the last 50 years, represented the mainstay of pharmacological treatment for the rheumatic diseases. However, the deleterious side effects associated with the use of many of these synthetic drugs (Lichtenstein D R, Syngal S, Wolfe M M. Nonsteroidal antiinflammatory drugs and the gastrointestinal tract. The double-edged sword. Arthritis Rheum 1995;38:5-18, Davies M N, Wallace J L. Nonsteroidal anti-inflammatory drug-induced gastrointestinal toxicity. New insights into an old problem. J Gastroenterol 1997;32:127-33, Manoukian A V, Carson J L. Nonsteroidal anti-inflammatory drug-induced hepatic disorders. Incidence and prevention. Drug Safety 1996;15:64-71, Huskisson E C, Berry H, Gishen P, Jubb R W, Whitehead J. Effects of antiinflammatory drugs on the progression of osteoarthritis of the knee. J Rheumatol 1995;22:1941-6) has prompted the evaluation and development of alternative treatments, particularly remedies from edible plant and animal sources which, by their very nature, are expected to be free of adverse side-effects.
The most widely used products in this regard are glucosamine and chondroitin sulphate (ChS) which are both constituents of cartilage PGs. Although it should be noted that the glucosamine used commercially is generally isolated from the chitosan present in the exoskeleton of crustacea. Controlled clinical studies conducted with glucosamine and chondroitin sulfate, alone or in combination, have indicated that they can provide relief of symptoms in OA (McAlindon T E, LaValley M P, Gulin J P, Felson D T, Glucosamine and Chondroitin for the treatment of Osteoarthritis: a systematic quality assessment and meta-analysis. JAMA, 2000; 263: 1469-1475). These agents have been categorised as slow acting disease modifying anti-osteoarthritis nutraceuticals. We are also aware of a number of patent disclosures describing the use of these agents alone and in combination with various other medicants for the treatment of OA and other musculoskeletal disorders (U.S. Pat. No. 5,364,845, Nov. 15, 1994; U.S. Pat. No. 6,136,795, Oct. 24, 2000; U.S. Pat. No. 6,162,787, Dec. 19, 2000, U.S. Pat. No. 6,271,213, Aug. 7, 2001, U.S. Pat. No. 6,432,929, Aug. 13, 2002, and references cited therein).
Since the ChSs are obtained from natural sources they can be sold directly to the public as food additives or supplements and are not presently require to comply with the rigorous quality control criteria used for synthetically manufactured pharmaceuticals as required by government agencies such as the FDA. Commercially available chondroitin sulfates are normally manufactured from bovine tissues such as lung and trachea by hydrolysis of the GAG protein core linkage of the cartilage PGs using either chemical or enzymatic procedures (U.S. Pat. No. 1,950,100 March 1932, Australian Patents AU-A1-66307/80 January 1981, AU-A-70540/87 December 1987, U.S. Pat. No. 6,162,787, Dec. 19, 2000 and references cited therein). The negatively charged water soluble ChS may be separated and purified from the proteins and peptides also generated by the hydrolysis of cartilage by multiple precipitations with acetone, aliphatic alcohols or the formation of water insoluble complexes with quaternary ammonium salts such as cetyl pyridinium chloride (CPC) (U.S. Pat. No. 1,950,100 March 1932, Australian Patents AU-A1-66307/80 January 1981, AU-A-70540/87 December 1987). However, none of these methods readily remove the contaminating nucleic acids (DNA and RNA) and other intracellular components also released during the chemical or enzymatic disruption of cartilage since these macromolecules are also anionic and would co-precipitate with the anionically charged ChS.
Contaminating nucleic acids could be selectively removed from the ChS by digestion with enzymes which degrade these contaminating molecules (eg, ribonucleases such as Benzonase (Mercke)), however this is an expensive procedure and the enzymes used would still have to be removed from the ChS preparation at some stage. Choatropic solvents such as guanidine hydrochloride and salt solutions of high ionic strength, such as potassium chloride have also been previously used to extract native proteoglycans from cartilaginous tissues but at low temperatures (below 4 degrees C.) and with the addition of protease inhibitors to prevent degradation of the required macromolecules by endogenous enzymes (Hascall V C and Sajdera S W, Protein-polysaccharide complex from bovine nasal cartilage. The function of glycoprotein in the formation of aggregates. J. Biological Chem. 1969,244;2384-2396; Oegema T, Hascall V, Dziewiatkowski D, Isolation and characterisation of proteoglycans from the rat chondrosarcoma. J. Biol. Chem. 1975, 250: 6151-6159; Inerot S and Heinegard D, Bovine tracheal cartilage proteoglycans. Variations in structure and composition with age. Collagen and Related Research, 1983, 3: 245-262). However, in order to release the GAG-peptides or ChS chains from these so isolated proteoglycan complexes it is necessary to subject them to proteolytic digestion by the addition of exogenous enzymes such as papain to degrade the their protein core (U.S. Pat. No. 6,162,787, Dec. 19, 2000, Inerot S and Heinegard D, Bovine tracheal cartilage proteoglycans. Variations in structure and composition with age. Collagen and Related Research, 1983, 3: 245-262, and references cited therein). Furthermore, use of these high ionic strength or chaotropic conditions to extract cartilage also disrupts cell membranes and thus release intra-cellular components, such as nucleic acids into the aqueous medium along with the PGs.
Uncharacterised GAG-peptides have also been prepared from bovine nasal cartilage but not bovine tracheal cartilage using sodium acetate or water at pH 4.5 (Nakano T. Nakano K, Sim J S, Extraction of glycosaminglycan peptide from bovine nasal cartilage with 0.1 M sodium acetate, J Agriculture and Food Chemistry, 1998, 46; 772-778, Nakano T, Ikawa N, Ozimek L, An economical method for the extraction of chondroitin sulfate-peptide from bovine nasal cartilage. Can Agric Engineering, 2000, 42; 205-208). However, neither the presence or absence of DNA in these GAG-peptide preparations nor their respective pharmacological activities nor direct use for the treatment of musculoskeletal conditions described could be inferred from the information recorded in these publications. Again, in the Nakano et al disclosures subsequent proteolytic or chemical hydrolysis of the products isolated by this method of extraction was necessary to obtain the ChS ultimately required by the authors.
In this regard it is important to note that most of the proteolytic enzymes used to exhaustively digest connective tissues to manufacture products of commercial interest, such as the ChSs are derived from bacterial or plant sources because of their broad range of substrate specificity and widespread availability. The amino acid sequences which are recognised and cleaved by these enzymes, as well as the amino acid sequences of the polypeptide fragments generated by their proteolytic actions, are therefore different to the sites of cleavage and polypeptide sequences produced by the endogenous proteinases of mammalian connective tissues. For example, studies with the mammalian class of cysteine proteinases, the Cathepsins, have shown that their preferred substrate binding and catalytic cleavage sites are different from that of the plant derived cysteine proteinase, papain (Barrett A j, Buttle D J, Mason R w, Lysosomal cysteine proteinases, ISI Atlas of Science, 1988: 256-260). In addition, while digestion of purified preparations of PGs with papain released single ChS chains with about 10 amino acid stubs still glycosidically attached the corresponding digestions of cartilage PGs with the cathepsins, D or B or G produced clusters containing 2 or more ChS chains and longer amino acid stubs with amino acid sequences different to those generated by the papain digested PGs (Roughley P J and Barrett A J, The degradation of cartilage proteoglycans by tissue proteinases, Biochem J, 1977,167: 629-637).
Certain patent disclosures cite methods of preparation and use of hydrolysates of cartilage for the treatment of musculoskeletal disorders and joint cartilage defects but these inventions are limited to the use of the peptides produced from type I and type II collagens for such treatments and make no reference to pharmacological activities of any GAG-peptide complexes when used alone or in combination with these collagen derived polypeptides. Furthermore none of these previous disclosures recognises the absence or presence of intra-cellular contaminants such as nucleic acids in their preparations (U.S. Pat. No. 3,966,908, Jun. 29, 1976, U.S. Pat. No. 4,804,745 February 1989, U.S. Pat. No. 5,399,347 March 1995, U.S. Pat. No. 5,364,845 December 1996, U.S. Pat. No. 6,025,327 February 2000, U.S. Pat. No. 6,372,794 April 2002).
While the consequences of long term human consumption of bovine or other animal nucleic acids in commercial ChS preparations sold as nutraceuticals or food supplements is presently unknown, it should be noted that these intracellular anionic macromolecules are strongly bound to or form complexes with retroviruses and heat/protease resistant prion proteins which have been implicated in the spread of transmissible spongiform encephalopathies such as Creutzfeld-Jakob disease, kuru, (Gerstmann-Straussler-Scheiner syndrome in humans, scrapie in sheep and goats, and bovine spongioform encaphalopathies in cattle. (Weissmann C et al, Transmission of prions, www.pnas.org/cgi/doi/1073/pnas. 172403799; Narang H, A critical review of the nature of the spongiform encephalopathy agent: protein theory versus virus theory, Exp Biol. Med, 2002,227: 4-19; C, et al, The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein, J. Mol. Biol., 2001, 307: 1011-1021; Nandi P K and Sizaret P-Y, Murine recombinent prion protein induces ordered aggregation of nucleic acids to condenced globular structures, Arch Virol, 2001, 146: 327-45; Cominicini S, et al, Genomic organisation, comparative analysis and genetic polymorphisms of the bovine and ovine prion Doppel genes (PRND), Mamm Genome 2001, 9: 729-33,). These intracellular entities could therefore be consumed by the subject in appreciable amounts when they comply with the manufactures recommended dosage of one or more grams of ChS daily for the suppression of the symptoms arising from osteoarthritis and related conditions.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.